Brucellae are highly infectious intracellular pathogens found in a wide range of mammals causing abortion, infertility and undulant fever. Unlike other intracellular pathogens, Brucella spp. do not encode classical virulent factors. However, chronic intracellular persistence in host cells indicates Brucella effectors likely interact with unidentified eukaryotic targets to modify the cellular environment for bacterial survival. Minimal information is available on Brucella proteins that contribute to bacterial virulence indicating an immediate need to explore the mechanisms of Brucella pathogenesis. This proposal aims to characterize the contribution of a newly identified Brucella protein, termed TcpB encoded by BMEI1674 that contains a TIR domain and mimics properties of the Toll-like receptor adaptor protein TIRAP. TcpB efficiently blocks TLR2 and TLR4 mediated NF-?B activation and plays a significant role in the virulence of the bacterium. Our studies indicated that TcpB interacts with phosphatidylinositols (PtDIns) and co-localizes with the plasma membrane and cytoskeleton network. We hypothesize that TcpB-PtDIns interaction provides TLR specificity for TcpB, but the significance of TcpB-microtubule interaction, mediated by the BB-loop motif of TcpB is unknown. The exact mechanism of TcpB mediated NF-?B suppression is not yet understood, and no information is available on the secretion of TcpB by Brucella and the escape of TcpB from Brucella containing vacuoles. Based on our preliminary studies, TcpB plays a broader role in addition to the suppression of innate immune response. Therefore the present proposal will characterize TcpB in detail to unravel the mechanisms of action and bacterial secretion. The specific aims of the present proposal are: Aim 1. To explore the detailed mechanism of TcpB mediated NF-?B suppression. Large-scale yeast-two hybrid screening will analyze TcpB interaction with unidentified mammalian proteins. Positive interactions will be confirmed by co-immunuprecipitation and co-sub-cellular localization experiments. Aim 2. To analyze the secretion of TcpB by Brucella melitensis and membrane penetration of TcpB. Secretion of TcpB by Brucella.melitensis will be analyzed in culture medium as well as in host cells. Western analysis of concentrated culture supernatant will be performed to test the secretion of TcpB. To analyze the secretion of TcpB inside the cells, a peptide tag-based (GSK-tag) reporter system over expressing TcpB-GSK will be used. To analyze the cell permeability purified TcpB protein will be incubated with mammalian cells and intracellular localized TcpB will be determined by western blotting. A cytokine ELISA will confirm that TcpB can enter host cells as well as demonstrate the suppression of cytokine by intracellular TcpB protein. Aim 3: To analyze the effect of TcpB on tubulin polymerization and TcpB mediated activation of small GTPases. To analyze the effect of TcpB on kinetics of tubulin assembly, fluorescent-based tubulin polymerization assays will be performed in the presence of purified TcpB or peptide harboring the BB-loop motif of TcpB that mediates microtubule interaction. The role of intact microtubules for TcpB mediated NF-?B suppression will be analyzed by reporter assays in the presence of the microtubule destabilizing agent nocodazole. To investigate the TcpB mediated activation of small GTPases, GTPase pull-down assays will be performed with the cell lysate transfected with TcpB. Phagocytosis assays will be performed to address whether TcpB mediated membrane ruffles and lamellipodia protrusions enhances phagocytosis. Our Preliminary Studies have identified a B. melitensis TIR domain containing protein, termed TcpB that possesses the ability to alter signaling events in host cells and disrupt innate immune mechanisms. Critical information gained from these specific aims will begin to bridge the gap between what we know about B. melitensis virulence and how virulence is regulated.